DNA contains the basic blueprint of almost all living organisms, and understanding the information of DNA molecules is a crucial topic for biological research and biomedical engineering. However, in a bulk solution, DNA molecules are typically coiled up, which can make analysis difficult. To reveal the genomic information of DNA molecules, various approaches have been investigated to stretch the tangled DNA molecules, such as optical and magnetic tweezers, surface-based combing, and extension in hydrodynamic flow.
DNA linearization is the prerequisite and critical step in single-molecule DNA analysis. When confined in a nanochannel, a coiled DNA molecule naturally stretches out as a combined result of its elastic properties and the excluded volume effect, depending on the dimensions of the nanochannel. Compared with other competing approaches, DNA stretching by nanoconfinement allows for a uniform elongation where the confined DNA molecule is exposed to the same confinement force and provides for long observation time of DNA in its unraveled state. The feature size of nanofluidic devices for DNA manipulation are usually required to be comparable or smaller than the persistence length of double-strand DNA (i.e., on the order of 10 to 100 nanometers) and uniform over tens to hundreds of micrometers and even centimeters. Conventional methods for patterning sub-100 nm channels use scanning beam lithography, where a beam is scanned across a substrate, such as in electron beam lithography (EBL) and focused ion beam lithography (FIB). However, conventional nanolithography techniques are usually very expensive and time consuming, and the devices are generally not reusable once contaminated. Alternative non-lithographic methods based on nanocracking can reduce the cost of device, but the uniformity of the resulting nanochannel arrays is relatively low. Also, the step interface between the loading microchannels and the stretching nanochannels in conventional nanofluidic devices poses a large entropic barrier, where the coiled DNA molecules clog at the junction. Although gray-scale lithography can harness this entropic force by creating a gradient at the interface, the mask design process is complicated and the fabrication of a refined gradient structure remains challenging. A convex lens-induced confinement (CLIC) platform can utilize the curved surface of a convex lens to locally deform a flexible coverslip above the micro/nanostructured surface, but expensive EBL is still needed to produce nanochannels on the glass surface of the CLIC platforms, and the depth of the generated nanochannels are not uniform. In addition, the nanochannels in conventional nanofluidic devices are essentially directly bonded. High pressure or electric fields are required for surface passivation, sample transport, and buffer refreshment due to the high hydraulic resistance of the nanochannels.
Existing DNA analysis methods and devices have many problems, and several challenges remain in order to reduce the cost of nanofluidic devices and improve the performance for DNA analysis with such devices.